Abstract
Alternative pre-mRNA splicing is a critical step to generate multiple transcripts, thereby dramatically enlarging the proteomic diversity. Thus, a common feature of most alternative splicing factor knockout models is lethality. However, little is known about lineage-specific alternative splicing regulators in a physiological setting. Here, we report that NSrp70 is selectively expressed in developing thymocytes, highest at the double-positive (DP) stage. Global splicing and transcriptional profiling revealed that NSrp70 regulates the cell cycle and survival of thymocytes by controlling the alternative processing of various RNA splicing factors, including the oncogenic splicing factor SRSF1. A conditional-knockout of Nsrp1 (NSrp70-cKO) using CD4Cre developed severe defects in T cell maturation to single-positive thymocytes, due to insufficient T cell receptor (TCR) signaling and uncontrolled cell growth and death. Mice displayed severe peripheral lymphopenia and could not optimally control tumor growth. This study establishes a model to address the function of lymphoid-lineage-specific alternative splicing factor NSrp70 in a thymic T cell developmental pathway.
INTRODUCTION
Nuclear speckles are irregular and non-membranous punctuate structures that serve as storage/assembly sites for pre-mRNA splicing machinery, and therefore regulate gene expression (1). Proteins belonging to the nuclear speckles include small nuclear ribonucleoprotein particles (snRNPs) and many non-snRNP splicing factors such as SR (serine/arginine) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) (2). Previously, our laboratory discovered a new SR-related protein named nuclear speckle-related protein 70 (NSrp70), which co-localizes and interacts physically with SRSF1 and SRSF2 in nuclear speckles through its RS-like region. NSrp70 modulates alternative splicing site selection of some mRNAs, including Fas v6, Tra2β1 v2 and CD44 exon v5 (3). However, little is known about the significance of NSrp70 in a physiological setting. One clue is that, like other major splicing factors, a knockout of NSrp70 resulted in an embryonic lethal phenotype (3), suggesting that this protein is essential for early development. Interestingly, NSrp70 is predominantly expressed in immune function-related tissues or organs (3), suggesting its potential significance in the immune system. This fact led us to investigate whether NSrp70 plays a role as a lineage-selective splicing regulator in the immune system.
The immune system is where rapid cell division and complex developmental processes occur during hematopoiesis. Notably, T cell development in the thymus is a highly integrated process with distinct developmental stages defined by the expression of the co-receptors CD4 and CD8 (4). These events are primarily driven by the expression and activation of the T cell receptor (TCR) complex. In particular, precise control of TCR signaling is critical for complete thymocyte development (5–7). Further, many factors that regulate the cell cycle, apoptosis, survival, differentiation, and proliferation are also cooperatively involved in thymocyte development (8–11).
A previous report demonstrated that conditional deletion of the prototypical SR protein SRSF2 (also known as SC35) in the thymus causes a defect in T cell maturation (12). An hnRNP L knockout resulted in decreased thymic cellularity caused by a partial block at the transition between DN4 and DP thymocytes and higher proliferation rates in DN4 thymocytes (13). However, the actions of SRSF2 and hnRNP L are not restricted to lymphocytes; indeed, they impact almost all biological processes (14). To our surprise, NSrp70 was highly and selectively detected in developing lymphocytes—highest in DP thymocytes—, strongly suggesting that NSrp70 may have lymphocyte-selective activity during the developmental process.
To address a potential role of NSrp70 in T cell development, we created a conditional-knockout using the CD4Cre-loxP strategy. NSrp70 deletion (Nsrp1f/fCD4Cre) profoundly perturbed the late development of DP thymocytes, leading to a significant reduction of CD4+ and CD8+ single positive (SP) cells in the thymus and peripheral lymphoid tissues. Global splicing profiling revealed that NSrp70 controls the processing of cell cycle-related RNA splicing factors, including Srsf1, Srsf7 and Son (15–17), and spliceosome-related factors, including Sf3b1 and U2af2 (18). NSrp70 also organizes the nuclear speckles by physically integrating the RNA splicing proteins and governs the cell cycle and survival at the stage of TCR-mediated positive selection to produce CD4+ or CD8+ SP T cell populations.
